Protein A-silica immunoadsorbent and process for its production

ABSTRACT

An immunoadsorbent material for removing IgG and IgG-complexes from biological fluids is prepared by covalently binding protein A to a solid-phase silica matrix. It has been found that particularly stable, high-capacity immunoadsorbents are obtained by derivatizing the silica with amino and/or carboxyl groups, and reacting the protein A with a carbodiimide at a pH in the range from 3.5 to 4.5. Binding through free hydroxyl groups may be achieved with cyanogen halides at a pH in the range from 11.0 to 11.5. After acid washing (pH 2.0-2.5) to remove non-covalently bound protein A, the immunoadsorbent may be employed in a column for therapeutic treatment of various cancers and autoimmune disorders where IgG-complexes are implicated as suppressing factors in inhibiting a normal immune response.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The extracorporeal treatment of blood to remove immunoglobulins andcirculating immune complexes may be useful in a variety ofcircumstances. For example, it is suspected that some cancer patientsdevelop a particular immune complex consisting of the patient's own IgGand an antigen associated with the cancer. It is thought that suchcomplexes can interfere with the functioning of the patient's immunesystem and prevent the immune system from responding to the cancer. Asystem for adsorbing IgG can be used to remove the IgG complexes fromthe blood, allowing the patient's natural immune defenses to resumetheir proper function. In addition, a variety of "autoimmune" disordersinvolve the production of antibodies which are specific for thepatient's own body. Severe harm can arise from such a misdirected immuneresponse, causing illness and even death to the patient.Immunoadsorption of the antibodies may protect the body from furtherdamage.

For these reasons, it is desirable to provide a system to facilitateremoving antibodies from a patient's blood. It is particularly desirablethat the device be convenient to use, sterile, and avoid the release oftoxic substances into the blood being treated.

2. Description of the Prior Art

Heat and formalin-treated Staphylococcus aureus Cowan I packed in acolumn has been employed for the removal of IgG from blood. See, e.g.,Jones et al. (1980) Cancer 46:675-684; Ray et al. (1980) Cancer45:2633-2638; and Holohan et al. (1982) Cancer Res. 42:3663-3668. Such asystem suffers from a number of disadvantages. Flow rate of plasmathrough the column is slow, and the column is subject to clogging.Moreover, possibly toxic bacterial cell wall components may be leachedinto the blood during the perfusion process. An improved system isdescribed by Terman et al. (1981) N. Engl. J. Med. 305:1195-1200.Protein A is entrapped within a charcoal matrix and utilized to treatplasma. The system, however, is difficult to sterilize and it appearsthat there is significant loss of the protein A into the patient's blood(Balint et al. (1984) Cancer Res. 44:734-743). Blood treatment systemsfor the removal of anti-A and anti-B antibodies are described byBensinger et al. (1981) N. Engl. J. Med. 304:160-162 and Bensinger etal. (1982) J. Clin. Apheresis 1:2-5. The immunoadsorption systemutilizes synthetic human blood group antigens covalently linked to asilica matrix. The use of a protein A-silica column for extracorporealimmunoadsorption is briefly reported by Bensinger et al. (1982) N. Engl.J. Med. 306:935. "Affinity Chromatorgraphy--Principles and Methods"published by Pharmacia Fine Chemicals, Uppsala, Sweden, teaches thatcarbodiimide coupling to Sepharose® is best performed at a pH above 4.5.

SUMMARY OF THE INVENTION

The present invention provides a method for preparing an immunoadsorbentmaterial and a system employing the immunoadsorbent material for theextracorporeal removal of IgG and IgG-complexes from biological fluids,such as blood and plasma. Such treatment may be useful for the treatmentof various autoimmune disorders as well as treatment of certain cancerswhere the presence of immunoglobulin complexes in the blood appears toinhibit the patient's immune response to the cancer. Additionally, themethod and system will find use in treating plasma to remove undesiredimmunoglobulins under a variety of circumstances, e.g., prior totransfusion.

The system of the present invention utilizes an immunoadsorbent materialwhich comprises purified protein A covalently coupled to a solid-phasematrix under particular conditions. The solid-phase matrix is either anamorphous or crystalline silica, and covalent coupling is accomplishedby appropriately derivatizing the solid-phase matrix and linking theprotein under conditions which have been found to maximize the bindingactivity and capacity of the resulting immunoadsorbent material. Theimmunoadsorbents thus formed have a very high capacity for adsorption ofthe immunoglobulins, are highly stable and avoid release of the bindingproteins into the biological fluid, are non-toxic, and are resistant tothe generation of fines during subsequent handling. Additionally, theadsorbent thus prepared can be air-dried, loaded into a biocompatiblecartridge, and gas sterilized. The device can then be transported in asterile condition, rehydrated on site, and employed for clinicaltreatment in a one-use disposable manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view of the immunoadsorbent column of the presentinvention.

FIG. 2 is a diagrammatic representation of a system for theextracorporeal treatment of blood constructed according to the presentinvention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

An immunoadsorbent column having a novel immunoadsorbent materialtherein is provided for the extracorporeal treatment of a biologicalfluid, such as plasma, to remove IgG and IgG-complexes therefrom. Thetreatment may be provided by continuously removing a patient's blood,separating the blood cells therefrom, treating the separated plasma inthe immunoadsorbent column to remove the IgG and IgG-complexes, andmixing and returning the treated plasma and blood cells directly to thepatient. Alternatively, after the blood has been removed and the bloodcells separated, the blood cells may be directly reinfused into thepatient. The separated plasma may be collected, treated in theimmunoadsorbent column of the present invention, again collected andthen returned to the patient as early as possible.

The novel immunoadsorbent material of the present invention comprisesprotein A covalently coupled to a solid-phase silica matrix underparticular conditions which have been found to maximize activity of theprotein A and binding capacity of the column while minimizing leakage ofthe protein A and other substances from the column during use.

Protein A is cell surface protein which is isolated from particularstrains of Staphylococcus aureus and able to bind free IgG andIgG-complexes. IgG-complexes are antigen-IgG complexes which circulatein patient serum and are not removed by the normal phagocytic mechanismsof the immune system. As stated above, removal of such circulatingIgG-complexes is useful in the treatment of a variety of disorders,including autoimmune disorders and cancer. The immunoadsorbent materialof the present invention will have a binding capacity of at least 5 mgIgG/gm adsorbent, usually 7 mg/gm or greater. The immunoadsorbent systemof the present invention allows removal of up to about 750 to 1500 mg ofthe circulating IgG-complexes, usually about 1000 mg by treatment of theplasma.

Protein A may be obtained from cultures of Staphylococcus aureus, forexample S. aureus Cowan I, by harvesting the cells and lysing with asuitable lytic agent, such as lysostaphin. The protein A may then bepurified by any suitable technique, such as ion exchange combined withmolecular sieve chromatography, to a final purity of 90-99%, usuallyabout 95%. Alternatively, suitably purified protein A may be obtainedfrom a number of commercial suppliers, such as IMRE Corporation,Seattle, Wash.

The solid-phase silica matrix may comprise virtually any form ofparticulate silica including amorphous silicas, such as colloidalsilica, silica gels, precipitated silicas, and fumed or pyrogenicsilicas; microcrystalline silicas such as diatomites; and crystallinesilicas such as quartz. The silica should have a particle size in therange from about 45 to 120 mesh, usually in the range from 45 to 60mesh.

In the preferred embodiment, the solid-phase matrix of theimmunoadsorbent material will be formed from diatomite aggregates.Usually, the diatomite material will be calcined to remove any remainingorganic material and to harden the surface of the aggregates in order tolessen breakage and degradation of the immunoadsorbent during use. Thediatomite material will consist primarily of silica (silicon dioxide)with lesser amounts of other minerals, including aluminum oxide, calciumoxide, magnesium oxide, ferric oxide, and the like. Usually, thediatomite material will comprise at least 80% silica, with less than 5%by weight of any other mineral. Other impurities may be present in thediatomite, but care should be taken that such impurities are non-toxicand non-degradative to the biological fluid being treated. Aparticularly suitable solid-phase silica (diatomite) matrix may beobtained from Johns-Manville Corporation under the tradenameChromosorb®.

The protein A is covalently coupled to the solid-phase silica matrix byderivatizing the matrix to introduce active reactive functional groups,and reacting the derivatized matrix with a coupling agent or underchemical conditions which binds the protein A to the matrix. Exemplaryprotocols for such binding are as follows.

Amino groups may be introduced to the silica matrix as the reactivefunctional group by any suitable method. For example, the silica matrixis first acid washed, followed by extensive rinsing with water anddrying. The acid washed silica is then reacted in a 5% to 10% solutionof an aminosilane, such as γ-aminopropyltriethoxysilane, with the pHadjusted to about 3.0. After 2 hours at about 75° C., the silica matrixis again washed extensively with water and dried overnight at 100° C.

Carboxyl groups may be introduced to the silica matrix as the reactivefunctional group by further reacting the amino-derivatized material, asjust described, with succinic anhydride as follows. The silica matrix ismixed with succinic anhydride in a suitable buffer, such as 0.5 Mphosphate buffer, and the pH adjusted to about 6.0. After 12 to 16 hoursat room temperature, the silica matrix is extensively washed, and dried.

Hydroxyl group (in addition to those hydroxyl groups occurring in thenative structure of the matrix) may be introduced to the silica matrixby any suitable method. For example, the silica matrix is first acidwashed, rinsed extensively with water, and dried. The acid washed silicais then reacted in a 5-10% solution of a silane such asγ-glycidoxypropyltrimethoxysilane. After a 2 hour incubation at 75° C.,the silica matrix is again washed extensively with water and dried at100° C.

Once the silica matrix has been derivatized with either amino and/orcarboxyl groups, the protein A is introduced by reaction with acarbodiimide which forms a covalent link between the matrix and theprotein A. The carbodiimide will have the formula:

    R'--N═C═N--R"

where R' and R" may be the same or different, being either alkyl,substituted-alkyl, benzyl, substituted-benzyl, or hydrogen. Alkyl orsubstituted-alkyl may be straight, branched or cyclic, and R willusually have fewer than 16 atoms other than hydrogen, more usually fewerthan 12 atoms, and six or fewer heteroatoms (i.e., other than carbon andhydrogen). If substituted-benzyl, R will usually have three or fewersubstitutions which will typically be halogen atoms. Suitablecarbodiimides are well known in the art. The preferred carbodiimide is1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate.

The binding reaction for the amino-derivatized matrix is carried outunder the following conditions. The protein A is mixed in water in thepresence of the carbodiimide. The pH of the solution is adjusted to therange from 3.5 to 4.5, usually about 3.5, and the silica matrix isintroduced and gently mixed for an extended period, usually about 5 to30 hours, more usually about 20 to 25 hours at room temperature. Thematrix is then extensively washed with water, dried, and acid washed ata pH from about 2.0 to 2.5, usually about 2.25, to remove labile proteinand other substances which are non-covalently bound to the silicamatrix. The material is then finally washed, dried and checked for thepresence of pyrogens. A suitable test for the presence of pyrogens isthe limulus ambeocyte lysate (LAL) test, commercially available as a kitfrom Marine Biologicals, Inc., P. 0. Box 546, Marmora, N.J. 08222.

The binding process for the carboxyl-derivatized silica matrix is asfollows. A carbodiimide (as above) is dissolved in water, and thesolution is adjusted to a pH in the range from 3.5 to 4.5, usually about3.5 pH. After introducing the silica matrix, the solution is gentlymixed for an extended period, usually about 10 to 25 hours, more usuallyabout 12 to 20 hours, at room temperature. The silica matrix is thenremoved and extensively washed with water. The protein A is thendissolved in water, the pH adjusted to the range from 3.5 to 4.5,usually about 3.5, and the silica matrix added and mixed for about 15 to30 hours, usually about 20 to 25 hours at room temperature. The silicamatrix is then extensively washed with water, dried, and washed one timein an acid wash (pH 2.0 to 2.5, usually about 2.25) to removenon-covalently bound protein A and other substances. The silica matrixis then washed a final time, and checked for pyrogens.

The binding process for the hydroxyl derivatized silica matrix is asfollows. Cyanogen bromide is dissolved in water. The silica matrix isadded to water and the pH is adjusted to 11.0. The cyanogen bromidesolution is added to the silica matrix, the mixture is constantlystirred keeping the silica particles in suspension, and the pH ismaintained between 11.0 and 11.5 by addition of NaOH until pHstabilization occurs. The activated silica matrix is extensively washedwith water, mixed with a solution of protein A with the pH adjusted to8.5-9.0, and mixed overnight at 25° C. After coupling, the matrix iswashed extensively with water, dried, and washed one time in an acidwash, pH 2.5, to remove non-covalently bound and acid labile protein Alinkages. The silica matrix is washed a final time and checked forpyrogens.

As demonstrated in the Experimental section hereinafter, the pH range offrom 3.5 to 4.5 for binding of the protein A to the amino and/orcarboxyl functionalities on the silica matrix is critical. Similarly,the binding of the protein A to the hydroxyl functionalities at a pH inthe range from 8.5 to 9.0 is also critical. The efficiency of bindingand the retained activity of the protein A both diminish as the pHdeviates outside of these narrow ranges. Moreover, it has been foundthat a mild acid wash with a pH in the range from about 2.0 to 2.5successfully removes non-covalently bound substances from the silicamatrix, particularly cleaving labile protein A linkages. The acidtreatment is thus important in achieving a stable immunoadsorbentmaterial which is able to retain the IgG and IgG-complexes bound withinthe column and avoid loss of protein A into the serum being treated.

Referring now to FIG. 1, the construction of a suitable cartridge 10 forcontaining the immunoadsorbent material as just described isillustrated. The cartridge comprises a cylinder 12, a pair of retainingscreens 14, and a pair of end caps 16. The end caps 16 each include aflange element 18 projecting from one surface thereof and a connectornipple 20 projecting from the other surface thereof. The connectornipple includes an axial passage 22 therethrough to define inlet/outletports through the end caps 16.

The cylinder 12 includes an annular groove 26 at each end thereof. Theflange element 18 on each end cap includes a mating ring 28 on the innercylindrical surface thereof, which mating ring engages the annulargroove 26 when the caps are placed over the end of the cylinder 12. Eachscreen 14 includes a gasket 30 around its circumference, which gasketserves as a sealing member between the end cap 16 and the cylinder 12when the cartridge 10 is assembled. To assemble the cartridge 10, afirst screen 14 is placed over one end of the cylinder 12, and an endcap 16 is fitted over the screen 14. The cylinder 12 is then filled withthe immunoadsorbent material as described above, and assembly of thecartridge completed by placing the remaining screen 14 and end cap 16 inplace.

The dimensions of the cartridge 10 are not critical, and will depend onthe desired volume of immunoadsorbent material. The volume of thecylinder 12 will typically range from about 50 to 500 cc, having adiameter in the range from about 4 to 8 cm and a length in the rangefrom about 5 to 10 cm.

A column 11 (FIG. 2) which comprises a cartridge 10 containing asuitable amount of the immunoadsorbent material prepared as describedabove, may be sterilized, typically with a gas sterilant such asethylene oxide, and either used immediately or sealed and stored forlater use.

Prior to use, the column 11 will be washed with normal saline followedby a wash with normal saline containing heparin or other suitableanti-coagulant such as anti-coagulant citrate dextrose (ACD). The column11 may then be connected to a cell separator 40 (FIG. 2) to receiveseparated plasma therefrom. The cell separator 40 may be a continuousflow cell separator, such as an IBM Model 2997, available from IBM,Armonk, N.Y., or may comprise a semi-permeable membrane which allowspassage of the plasma and blood proteins, but prevents passage of thecellular elements of the blood. In the case of a semi-permeablemembrane, a blood pump 42 will be required to pass the blood through themembrane. Suitable blood pumps include a tube and peristaltic pumpswhere the blood is isolated from the pumping machinery to preventcontamination. The blood will pass through the cell separator 40 at arate in the range from about 10 to 20 ml/min typically until a totalvolume of about 2 liters of blood have been passed. The blood cells aremixed with the plasma passing from the treatment column 11, and therecombined blood returned to the patient. Typically, a microfilter 44 isprovided at the outlet of the treatment column 11 to prevent passage ofmacroscopic particles which might be lost from the column 11.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL 1. Preparation of Immunoadsorbent Material

Acid washed silica matrix (Chromosorb® P, No. C5889, Johns-Manville,1.25 kilograms) was weighed out, divided into 4 parts, and added to fourFernback type flasks. The matrix was re-hydrated with water andvigorously shaken overnight in a gyrotary shaker at approximately 150rpm. After this procedure, the silica matrix was extensively washed withwater to remove generated fine particles. This procedure appeared tomake the shape of the silica matrix particles more uniform resulting inmatrix particles which generate few fines in later handling procedures.After washing, the silica matrix was added to an approximately 5-10%solution of appropriate silane, incubated for 2 hours at 75° C.,extensively washed with water, and baked dry at 115° C.

The dried silanized silica matrix (1 kilogram) was re-hydrated andextensively washed with water to remove generated fines. The silicamatrix was then mixed with 2 grams of protein A and 50 grams ofcarbodiimide (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate) and the pH of the mixture adjusted to 3.5. Ourstudies indicated that a pH range of 3.5-4.5 yields a higher percentageuptake of protein A versus a higher pH range (4.5-5.0) (77.42% uptakeversus 67.49%). A lower pH range, below 3.5 was avoided because ofpossible acid hydrolysis of protein A during the prolonged bindingincubation.

The mixture was gently rotated on a roller apparatus for 22 hours at 25°C. The silica matrix was then extensively washed with water, dried at37° C., and the uptake of protein A was determined. After drying, 3liters of acid water, pH 2.5, was added to the silica matrix, incubatedfor 5 minutes at 25° C., and the amount of protein A released from thematrix was determined. The matrix was extensively washed with water,dried, and the amount of protein A per gram of silica was determined.The results were as follows:

Bound Protein A . . . 1966 mg

Protein A released . . . 440 mg

Protein A/gm adsorbent. . . 1.5 mg

2. Use of Immunoadsorbent to Separate IgG and

IgG Complexes from Normal Human Serum

Immunoadsorbent prepared as described above was incubated with 2 ml ofnormal human serum for 5 minutes at 25° C. After incubation, the silicamatrix was washed with 100 ml of phosphate buffered saline (PBS), pH7.5. Bound proteins were eluted with 12.5 ml of PBS, pH 2.5, andneutralized to pH 7.5. The total protein eluted was determined to beapproximately 10 mg, as described by Lowry et al. (1951) J. Biol. Chem.193:265-272. The eluted protein was subjected to polyacrylamide gelelectrophoresis, and prominent bands were detected at 50 kD and 25 kD,corresponding to the heavy and light chains of IgG, respectively. Thepresence of IgG was confirmed by double immunodiffusion analysisemploying γ-chain specific anti-human IgG.

To determine removal of IgG complexes by the immunoadsorbent, 2.5 ml ofnormal human serum was incubated with heat-aggregated human IgG to fixcomplement to the aggregates. This combination behaves as immunecomplexed IgG. Immunoadsorbent prepared as described above was incubatedwith 0.8 ml of the heat-aggregated serum for 5 minutes at 25° C. Thiswas repeated 3 times until a total volume of 2.4 ml was passed throughthe immunoadsorbent, and fractions were collected until all the serumwas passed through the immunoadsorbent. IgG immune complexes in pre- andpost-perfusion serum fractions were measured employing the Raji cellbinding IgG immune complex assay as described by Theofilopoulos et al.(1974) J. Exp. Med. 140:1230-1244. The results are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Sample*   Immune Complex (μg/ml)                                                                       % Reduction                                       ______________________________________                                        Pre-perfusion                                                                           160               --                                                Post-perfusion                                                                Fraction 1                                                                              120               25                                                Fraction 2                                                                              125               22                                                Fraction 3                                                                              105               34                                                Fraction 4                                                                              104               35                                                ______________________________________                                         *Equivalent protein quantities were assayed to control for dilutional         effects.                                                                 

As shown in Table 1, immune complex leves of the serum were reduced bypassage through the immunoadsorbent.

3. Therapeutic Use of Column to Treat Kaposi's Sarcoma

A patient with advanced disseminated Kaposi's sarcoma and acquiredimmune deficiency syndrome (AIDS) was treated with plasma perfusion overa protein A column prepared as described above. The immunologic andtumor changes observed during the treatment are reported below.

The patient was a 44 year old homosexual man with a history of extensiveKaposi's sarcoma and AIDS. Skin lesions were first noted on the feet andlegs, with inguinal node involvement, 26 months prior to protein Atherapy. At that time, weekly intravenous treatments with vinblastinewere initiated; the dosage was adjusted according to the complete bloodcount. The Kaposi's sarcoma remained stable and regionalized to thelower extremities for 1 year, at which time a few scattered lesionsslowly became apparent on the forearms and both calves. Six monthslater, because of enlargement of inguinal nodes and a new pubic lesion,chlorambucil, 2 mg b.i.d., was added. Within 2 months, the nodesdecreased and skin lesions stabilized, and chlorambucil wasdiscontinued. Disease activity again began to accelerate 4 months priorto protein A therapy, with new lesions forming on the trunk, face, legs,feet, arms, and hands, along with confluence of old lesions on the righttibia, despite continued vinblastine infusions. Dyspnea and dry coughbecame apparent 3 months later, with an X-ray film showing new bilateralbasilar infiltrates. Open lung biopsy revealed extensive involvement ofthe lung with Kaposi's sarcoma. Additional skin lesions appeared almostdaily, and the patient's condition continued to deteriorate.

At the time of admission for extracorporeal protein A perfusiontreatment, the patient's white blood cell count was 7,500 cells/mm³,with a differential of 70% polymorphonuclear leukocytes, 3% bands, 11%lymphocytes, 14% monocytes, and 2% eosinophils. The hemoglobin level was8.3 g/dl, and the hematocrit 25.6%, with a platelet count of 87,000/mm³. The total protein level was 5.3 g %, the albumin 2.8 g % and the IgGlevel was 1,170 mg %. Circulating immune complex (IgG-complex), measuredusing the solid-phase Clq-binding assay, were 4.7 μg equivalents ofaggregated human gamma globulin (AHG). Complement levels were 55 mg %(C3) and 6 mg % (C4).

Immunofluorescent studies with monoclonal antibodies (Ortho-mune,Raritan, NJ) on circulating lymphocytes revealed 58% total T cells (T₃),12% helper/inducer T cells (T₄), and 44% suppressor/cytotoxic T cells(T₈), with a T₄ /T₈ ratio of 0.21.

Pulmonary function tests revealed a forced vital capacity of 2.46 L (51%of predicted) and forced expiratory volume, in 1 second, of 1.64 L (45%of predicted). Carbon monoxide diffusion capacity was 57% of normal. Thearterial blood gases on room air were as follows: Po₂, 62 mm Hg; Pco₂,41 mm Hg; HC0₃, 30.5 mEq/L; pH 7.48.

The extracorporeal immunoadsorption procedures were performed in theintensive care unit. A Swan-Ganz catheter was placed into the pulmonaryartery to monitor hemodynamic changes. A continuous-flow plasma-cellseparator (IBM 2997, Armonk, NY) was used to separate anticoagulatedblood into cellular components and plasma. The cellular components werereturned unprocessed. The plasma was perfused over a column containing200 mg protein A covalently bound to silica prepared as described above,and returned to the patient. The protein A was isolated from purecultures of Staphylococcus aureus Cowan I employing lysostaphindigestion. Protein A purity was determined by polyacrylamide gelelectrophoresis, and IgG binding capacity was determined. The protein Awas covalently coupled to silica, loaded into a biocompatible cartridge,and sterilized by exposure to ethylene oxide. Sterility was confirmedemploying strips impregnated with spores of Bacillus subtilis (RavenBiological Laboratory, Omaha, NE). In addition, studies revealed thatextensive washing of the column with 4 L of sterile, pyrogen-free water,immediately prior to use, resulted in a lack of detectable pyrogens(Limulus amoebocyte lysate, Pyrogent®; Mallinckrodt, Inc., St. Louis,MO). Each protein A treatment column had the capacity to bindapproximately 1.5 g IgG from plasma. Plasma flow rates were between 10and 20 ml/min. Three liters of plasma was perfused during eachprocedure. Three treatments were performed over 7 days on anevery-other-day schedule. The patient was treated three times withextracorporeal perfusion of plasma over protein A. Three days after thelast procedure the patient died of respitory distress and an autopsy wasperformed.

RESULTS

No major complications occurred during the treatment procedures. A milddrop (10-20 mm Hg) in systolic blood pressure was observed, along withsinus tachycardia up to 120 beats/min, but neither required therapy.Changes in body temperature were less than 1° C. The patient reportedpain in tumor lesions on his right lower extremity during the lastprocedure; otherwise, no lesional discomfort was noted. The patient'spulmonary status remained stable throughout each procedure and, overall,the treatments were well tolerated.

No new lesions of the skin appeared after initiation of treatment.Grossly, about 20% of the skin lesions showed a slight decrease in sizealong with central necrosis. Erythematous halos appeared around thoselesions and were apparent after the second treatment. Healing wasinitiated in a large, confluent ulcerated lesion of the right tibia. Nomeasureable change in adenopathy was noted. Prior to treatments,intraoperative examination of the thorax during open lung biopsyrevealed flat, indurated, hemorrhagic plaques involving the pleura, andwidespread reddish nodular lesions of the lung parenchyma. At autopsyexamination, 16 hours after the patient's death, those same pleuralareas appeared to have definite central umbilication; the lung was morehemorrhagic, with a decrease in nodularity. Histologically, pretreatmenttissue from the open lung biopsy revealed characteristic Kaposi'ssarcoma, with nodular, hemorrhagic, densely cellular infiltrates ofplump, spindled cells forming scant fascicles, and numerous abortivevascular spaces. Postmortem microscopic examination of the lung revealeda decrease in tumor cell density, reduction in nuclear size, andincreasing collagen deposition between the tumor cells. Similar changeswere observed in some of the responding skin lesions compared withpretreatment biopsy specimens.

Biopsies from skin tumor lesions taken prior to treatment showed nodeposits of IgM, IgG, or IgA, nor of C3 or C4, as determined by directimmunofluorescence on frozen sections. Biopsies taken after the lastprocedure from the same lesions showed C3 deposits, but no deposition ofimmunoglobulins.

The changes in immunologic parameters are shown in Table 2. Of interest,the IgG-complex level increased from 2.6 μg equivalents of AHG prior tothe third treatment to 6.5 μg equivalents of AHG 24 hours later.

                  TABLE 2                                                         ______________________________________                                                       First     Second    Third                                      Parameter      procedure procedure procedure                                  ______________________________________                                        IgG (mg %)     1.170     820       808                                        Immune complexes (IgG)                                                                       4.7       2.3       6.5                                        (μg equivalents of AHG)                                                    Complement (mg %)                                                                            55(C3)    Not done  74(C3)                                                    6(C4)               5.5(C4)                                    Antilymphocyte 1:16      None      None                                       antibody titer           detectable                                                                              detectable                                 T.sub.4 /T.sub.8 ratio                                                                       0.21      0.18      0.19                                       Inhibitory factors                                                                           1:16      None      None                                                                detectable                                                                              detectable                                 Circulating lymphocytes                                                                      825       962       720                                        (per mm3)                                                                     ______________________________________                                         All values are pretreatment. AHG, aggregated human gamma globulin,            measured as described by Hay et al. (1976) Clin. Exp. Immunol. 24:396-400                                                                              

There were no significant changes in hematologic values, except for adecrease of 46% in the platelet count over the treatment period.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for preparing an immunoadsorbentmaterial useful for removing IgG and IgG-complexes from biologicalfluids, said method comprising:introducing free amino or carboxyl groupsonto a silica matrix; reacting the silica matrix with purified protein Ain the presence of a carbodiimide at a pH in the range from 3.5 to 4.5to covalently link the protein A to the matrix through the amino orcarboxyl groups; and washing the silica matrix at a pH in the range from2.0 to 2.5 to remove loosely bound protein A from the matrix.
 2. Amethod as in claim 1, wherein amino groups are introduced onto thesilica matrix by reaction with a silane.
 3. A method as in claim 2,wherein the amino silane is γ-aminoproplytriethoxysilane.
 4. A method asin claim 1, wherein carboxyl groups are introduced by first reacting thesilica matrix with an amino silane to introduce amino groups, followedby reacting the silica matrix with succinic anhydride to replace theamino groups with carboxyl groups.
 5. A method as in claim 1, whereinthe silica matrix is amorphous silica.
 6. A method as in claim 5,wherein the amorphous silica is diatomite.
 7. A method as in claim 6,wherein the diatomite is sized from 45 to 60 mesh.
 8. A method as inclaim 1, wherein the protein A is present at at least 0.05 weightpercent of the silica matrix.
 9. An immunoadsorbent material useful forremoving IgG and IgG-complexes from biological fluids, said materialcomprising purified protein A covalently coupled to a silica matrixprepared by the method comprising:introducing free amino or carboxylgroups onto a silica matrix; reacting the silica matrix with purifiedprotein A in the presence of a carbodiimide at a pH in the range from3.3 to 3.7 to covalently link the protein A to the matrix through theamino or carboxyl groups; and washing the silica matrix at a pH in therange from 2.0 to 2.5 to remove loosely bound protein A from the matrix.10. An immunoadsorbent material as in claim 9, wherein amino groups areintroduced onto the silica matrix by reaction with a silane.
 11. Animmunoadsorbent material as in claim 10, wherein the amino silane isγ-aminoproplytriethoxysilane.
 12. An immunoadsorbent material as inclaim 9, wherein carboxyl groups are introduced by first reacting thesilica matrix with an amino silane to introduce amino groups, followedby reacting the silica matrix with succinic anhydride to replace theamino groups with carboxyl groups.
 13. An immunoadsorbent material as inclaim 9, wherein the silica matrix is amorphous silica.
 14. Animmunoadsorbent material as in claim 13, wherein the amorphous silica isdiatomite.
 15. An immunoadsorbent material as in claim 9, wherein theprotein A is present at at least 0.05 weight percent of the silicamatrix.
 16. A method for preparing an immunoadsorbent material usefulfor removing IgG and IgG-complexes from biological fluids, said methodcomprising:introducing free hydroxyl groups onto a silica matrix;activating the silica matrix in the presence of cyanogen bromide at a pHin the range from 11.0 to 11.5, and covalently linking the protein A tothe matrix at a pH in the range from 8.5 to 9.0; and washing the silicamatrix at a pH in the range from 2.0 to 2.5 to remove loosely boundprotein A from the matrix.
 17. A method as in claim 16, wherein hydroxylgroups are introduced onto the silica matrix by reaction with a silane.18. A method as in claim 17, wherein the silane isγ-glycidoxypropyltrimethoxysilane.
 19. A method as in claim 16, whereinthe silica matrix is amorphous silica.
 20. A method as in claim 19,wherein the amorphous silica is diatomite.
 21. An immunoadsorbentmaterial useful for removing IgG and IgG-complexes from biologicalfluids, said material comprising purified protein A covalently coupledto a silica matrix prepared by the method comprising:introducing freehydroxyl groups onto a silica matrix; activating the silica matrix inthe presence of cyanogen bromide at a pH in the range from 11.0 to 11.5,and covalently linking the protein A at a pH in the range from 8.5 to9.0; and washing the silica matrix at a pH in the range from 2.0 to 2.5to remove loosely bound protein A from the matrix.
 22. Animmunoadsorbent material as in claim 21, wherein hydroxyl groups areintroduced onto the silica matrix by reaction with a silane.
 23. Animmunoadsorbent material as in claim 22, wherein the silane isγ-glycidoxypropyltrimethoxysilane.
 24. An immunoadsorbent material as inclaim 21, wherein the silica matrix is amorphous silica.
 25. Animmunoadsorbent material as in claim 24, wherein the amorphous silica isdiatomite.
 26. An immunoadsorbent material as in claim 25, wherein thediatomite is sized from 45 to 60 mesh.